CN110128542B - PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application - Google Patents
PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application Download PDFInfo
- Publication number
- CN110128542B CN110128542B CN201810127224.7A CN201810127224A CN110128542B CN 110128542 B CN110128542 B CN 110128542B CN 201810127224 A CN201810127224 A CN 201810127224A CN 110128542 B CN110128542 B CN 110128542B
- Authority
- CN
- China
- Prior art keywords
- pth
- pet
- fusion protein
- detection reagent
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 110
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 110
- 238000001514 detection method Methods 0.000 title claims abstract description 80
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 230000000087 stabilizing effect Effects 0.000 claims abstract description 78
- 108090000445 Parathyroid hormone Proteins 0.000 claims description 192
- 239000000243 solution Substances 0.000 claims description 69
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000013612 plasmid Substances 0.000 claims description 30
- 108020004414 DNA Proteins 0.000 claims description 22
- 230000014509 gene expression Effects 0.000 claims description 22
- 238000003776 cleavage reaction Methods 0.000 claims description 21
- 230000007017 scission Effects 0.000 claims description 21
- 210000002966 serum Anatomy 0.000 claims description 21
- 239000004471 Glycine Substances 0.000 claims description 18
- 229930006000 Sucrose Natural products 0.000 claims description 17
- 239000005720 sucrose Substances 0.000 claims description 17
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 16
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 16
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 16
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 16
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 15
- 238000003908 quality control method Methods 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 150000001413 amino acids Chemical group 0.000 claims description 13
- 239000008363 phosphate buffer Substances 0.000 claims description 13
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Substances [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 13
- 210000004027 cell Anatomy 0.000 claims description 12
- 239000012894 fetal calf serum Substances 0.000 claims description 12
- 102000053602 DNA Human genes 0.000 claims description 11
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 11
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 11
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 11
- 241001465754 Metazoa Species 0.000 claims description 9
- 150000001720 carbohydrates Chemical class 0.000 claims description 9
- 239000003599 detergent Substances 0.000 claims description 9
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims description 8
- 229920000136 polysorbate Polymers 0.000 claims description 8
- 108020004705 Codon Proteins 0.000 claims description 7
- 239000000427 antigen Substances 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 238000003018 immunoassay Methods 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 241000283707 Capra Species 0.000 claims description 5
- 229920002307 Dextran Polymers 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 241001195348 Nusa Species 0.000 claims description 5
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 claims description 5
- 229940107187 fructooligosaccharide Drugs 0.000 claims description 5
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 4
- 239000004380 Cholic acid Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 4
- 239000002738 chelating agent Substances 0.000 claims description 4
- 235000019416 cholic acid Nutrition 0.000 claims description 4
- 229960002471 cholic acid Drugs 0.000 claims description 4
- 229960003964 deoxycholic acid Drugs 0.000 claims description 4
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 239000003755 preservative agent Substances 0.000 claims description 4
- 230000002335 preservative effect Effects 0.000 claims description 4
- 229940083575 sodium dodecyl sulfate Drugs 0.000 claims description 4
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 101710083262 Ectin Proteins 0.000 claims description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 3
- 239000012888 bovine serum Substances 0.000 claims description 3
- 101150065880 btuF gene Proteins 0.000 claims description 3
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 3
- 239000012091 fetal bovine serum Substances 0.000 claims description 3
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 3
- 101001135770 Homo sapiens Parathyroid hormone Proteins 0.000 claims description 2
- 101001135995 Homo sapiens Probable peptidyl-tRNA hydrolase Proteins 0.000 claims description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 102100036829 Probable peptidyl-tRNA hydrolase Human genes 0.000 claims 13
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims 1
- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 239000007788 liquid Substances 0.000 abstract description 8
- 238000012216 screening Methods 0.000 abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 2
- 239000000199 parathyroid hormone Substances 0.000 description 206
- 102000003982 Parathyroid hormone Human genes 0.000 description 179
- 229960001319 parathyroid hormone Drugs 0.000 description 150
- 239000000047 product Substances 0.000 description 24
- 238000000034 method Methods 0.000 description 20
- 239000012634 fragment Substances 0.000 description 18
- 239000000126 substance Substances 0.000 description 18
- 230000000694 effects Effects 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 13
- 230000015556 catabolic process Effects 0.000 description 12
- 238000006731 degradation reaction Methods 0.000 description 12
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 description 11
- 239000013604 expression vector Substances 0.000 description 11
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 10
- 239000011575 calcium Substances 0.000 description 10
- 229910052791 calcium Inorganic materials 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 238000009472 formulation Methods 0.000 description 9
- 230000004927 fusion Effects 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 239000012085 test solution Substances 0.000 description 7
- 238000007865 diluting Methods 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 210000000988 bone and bone Anatomy 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 210000002990 parathyroid gland Anatomy 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 238000005520 cutting process Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000000149 penetrating effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- DLZKEQQWXODGGZ-KCJUWKMLSA-N 2-[[(2r)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]acetic acid Chemical group OC(=O)CNC(=O)[C@@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KCJUWKMLSA-N 0.000 description 2
- LEOJISUPFSWNMA-UHFFFAOYSA-N ABEI Chemical compound O=C1NNC(=O)C=2C1=CC(N(CCCCN)CC)=CC=2 LEOJISUPFSWNMA-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 208000013038 Hypocalcemia Diseases 0.000 description 2
- 102000005431 Molecular Chaperones Human genes 0.000 description 2
- 108010006519 Molecular Chaperones Proteins 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 229930003316 Vitamin D Natural products 0.000 description 2
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000000705 hypocalcaemia Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000000849 parathyroid Effects 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical compound C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 235000019166 vitamin D Nutrition 0.000 description 2
- 239000011710 vitamin D Substances 0.000 description 2
- 150000003710 vitamin D derivatives Chemical class 0.000 description 2
- 229940046008 vitamin d Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 1
- NINQYGGNRIBFSC-CIUDSAMLSA-N Ala-Lys-Ser Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CO)C(O)=O NINQYGGNRIBFSC-CIUDSAMLSA-N 0.000 description 1
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 1
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- BTJVOUQWFXABOI-IHRRRGAJSA-N Arg-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCNC(N)=N BTJVOUQWFXABOI-IHRRRGAJSA-N 0.000 description 1
- ULBHWNVWSCJLCO-NHCYSSNCSA-N Arg-Val-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N ULBHWNVWSCJLCO-NHCYSSNCSA-N 0.000 description 1
- WIDVAWAQBRAKTI-YUMQZZPRSA-N Asn-Leu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O WIDVAWAQBRAKTI-YUMQZZPRSA-N 0.000 description 1
- UYCPJVYQYARFGB-YDHLFZDLSA-N Asn-Phe-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O UYCPJVYQYARFGB-YDHLFZDLSA-N 0.000 description 1
- HPNDBHLITCHRSO-WHFBIAKZSA-N Asp-Ala-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(O)=O HPNDBHLITCHRSO-WHFBIAKZSA-N 0.000 description 1
- LIVXPXUVXFRWNY-CIUDSAMLSA-N Asp-Lys-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O LIVXPXUVXFRWNY-CIUDSAMLSA-N 0.000 description 1
- PLOKOIJSGCISHE-BYULHYEWSA-N Asp-Val-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PLOKOIJSGCISHE-BYULHYEWSA-N 0.000 description 1
- 108090000312 Calcium Channels Proteins 0.000 description 1
- 102000003922 Calcium Channels Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 1
- IXFVOPOHSRKJNG-LAEOZQHASA-N Gln-Asp-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IXFVOPOHSRKJNG-LAEOZQHASA-N 0.000 description 1
- NTBDVNJIWCKURJ-ACZMJKKPSA-N Glu-Asp-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NTBDVNJIWCKURJ-ACZMJKKPSA-N 0.000 description 1
- WVYJNPCWJYBHJG-YVNDNENWSA-N Glu-Ile-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O WVYJNPCWJYBHJG-YVNDNENWSA-N 0.000 description 1
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000037147 Hypercalcaemia Diseases 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 102000028555 IgG binding proteins Human genes 0.000 description 1
- 108091009325 IgG binding proteins Proteins 0.000 description 1
- POMXSEDNUXYPGK-IHRRRGAJSA-N Leu-Met-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N POMXSEDNUXYPGK-IHRRRGAJSA-N 0.000 description 1
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- OWRUUFUVXFREBD-KKUMJFAQSA-N Lys-His-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O OWRUUFUVXFREBD-KKUMJFAQSA-N 0.000 description 1
- HVAUKHLDSDDROB-KKUMJFAQSA-N Lys-Lys-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O HVAUKHLDSDDROB-KKUMJFAQSA-N 0.000 description 1
- IOQWIOPSKJOEKI-SRVKXCTJSA-N Lys-Ser-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IOQWIOPSKJOEKI-SRVKXCTJSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- HMRAQFJFTOLDKW-GUBZILKMSA-N Ser-His-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O HMRAQFJFTOLDKW-GUBZILKMSA-N 0.000 description 1
- NIOYDASGXWLHEZ-CIUDSAMLSA-N Ser-Met-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O NIOYDASGXWLHEZ-CIUDSAMLSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- 206010041277 Sodium retention Diseases 0.000 description 1
- 241000519995 Stachys sylvatica Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- AIISTODACBDQLW-WDSOQIARSA-N Trp-Leu-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 AIISTODACBDQLW-WDSOQIARSA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000004097 bone metabolism Effects 0.000 description 1
- 230000010072 bone remodeling Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 125000003716 cholic acid group Chemical group 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000005115 demineralization Methods 0.000 description 1
- 230000002328 demineralizing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000008713 feedback mechanism Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 102000058004 human PTH Human genes 0.000 description 1
- 230000000148 hypercalcaemia Effects 0.000 description 1
- 208000030915 hypercalcemia disease Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000002357 osmotic agent Substances 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 108010027977 parathyroid hormone (3-84) Proteins 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 101150031139 pth gene Proteins 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008060 renal absorption Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a PTH fusion protein, a preparation method thereof, a detection reagent containing the PTH fusion protein, a kit and application. The PTH fusion protein comprises: a tag sequence; and a full-length amino acid sequence of PTH positions 1-84, wherein the tag sequence is located at the C-terminus or N-terminus of the full-length amino acid sequence of PTH positions 1-84. By using a full-length PTH fusion protein with a tag sequence, a more comprehensive binding region is provided upon binding to an antibody, thereby improving detection accuracy. On the basis, in the preferred embodiment of the present application, the stable solution of the full-length PTH fusion protein is obtained by selecting a specific kind of stable solution and screening by optimizing the content ratio of the components. The PTH fusion protein can exist in a liquid state at normal temperature and can stably exist for a long time in the stabilizing solution, so that the accuracy of clinical detection and detection is improved, and the convenience of detection is improved.
Description
Technical Field
The invention relates to the field of immunodetection reagents, in particular to a PTH fusion protein, a preparation method thereof, a detection reagent containing the PTH fusion protein, a kit and application of the PTH fusion protein.
Background
PTH (parathyroid hormone) is useful in diagnosing the etiology of hypercalcemia and hypocalcemia and in distinguishing between parathyroid related disorders, and in monitoring the efficacy of parathyroid related disorders. PTH is usually detected in combination with serum calcium, and both are very important for understanding the blood calcium balance and the feedback mechanism of parathyroid gland to serum calcium. If PTH is severely or chronically imbalanced with calcium, the physician will take therapeutic measures.
PTH is produced by 4 parathyroid glands located behind the thyroid gland. Normally, parathyroid glands secrete PTH into the blood circulation when hypocalcemia occurs in the body. PTH then raises blood calcium to normal levels in three ways, each of which promotes bone calcium release; promoting kidney activation of vitamin D (the activation of vitamin D can increase the absorption of calcium by intestinal tract); inhibit calcium excretion from urine (sodium retention and phosphorus excretion). When blood calcium is elevated, PTH levels decrease. When PTH is released from the parathyroid gland into the blood, it subsequently cleaves into fragments (its half-life is less than 5 minutes).
PTH is a peptide hormone consisting of 84 amino acids (also known as PTH (1-84)), which plays an important physiological role in the regulation of bone metabolism. The physiological functions of PTH are mainly to stimulate renal reabsorption of calcium, secretion of phosphorus and bone remodeling, leading to synthesis and decomposition. Clinically, if the secretion of PTH is too high, abnormal PTH concentration in blood may cause hyperthyroidism, thereby causing bone demineralization. If an active fragment of PTH acts on bone at a low or moderate fluid concentration for a long time, it stimulates bone formation and effectively treats osteoporosis by inducing synthesis in bone. PTH also has an effect on the cardiovascular system through cell membrane calcium channels. Studies have shown that the active center of PTH is a 1-34 amino acid peptide segment located N-terminally, which acts by binding to specific receptors in renal bone tissue.
PTH contains no cysteine, is very unstable in vivo, and is low in content, and its isolation and purification are difficult. Several reports have described methods for direct expression of PTH in E.coli. For example BreyeL and Morellle et al, attempt to express PTH directly in E.coli, but because of RNA and protein instability, the PTH produced in very limited yields (< 500. mu.g/L). In addition, Born et al achieved the expression of truncated PTH, primarily PTH (3-84) and PTH (8-84), using a multicopy plasmid of the pre-PTH cDNA, but with little PTH bioactivity. The other strategy is a method using fusion protein, but most of the prior disclosures are to prepare PTH (1-34) fusion protein, and the preparation process involves the steps of extraction, renaturation, enzyme digestion and the like of inclusion bodies, so that the preparation is relatively complicated. In addition, Forsberg adopts IgG binding protein as a fusion partner to express PTH (1-84) in Escherichia coli, and the final yield can reach 5-8 mg/L. The PTH products prepared by the recombinant expression method are all stored in a freeze-dried form.
PTH calibration products and quality control products in the current market can be stored for a long time only by adopting a freeze-drying mode, the freeze-drying production process is complex, the use needs to be re-melted, the PTH calibration products and the quality control products can be quickly used after re-melting, the PTH calibration products and the quality control products cannot be placed for a long time, and inaccurate experimental results are easily caused. In view of the above problems, no good solution is available at present.
Disclosure of Invention
The invention mainly aims to provide a PTH fusion protein, a preparation method thereof, a detection reagent containing the PTH fusion protein, a kit and application, so as to provide a PTH full-length fusion protein with better activity, thereby improving the accuracy of clinical detection of PTH antigen.
In order to achieve the above object, according to one aspect of the present invention, there is provided a PTH fusion protein comprising: a tag sequence; and a full-length amino acid sequence of PTH1-84, wherein the tag sequence is located at the C-terminus or N-terminus of the full-length amino acid sequence of PTH 1-84.
Further, the tag sequence is selected from any one of: MBP, NusA, TF, MysB, GST, T7, 6 His + T7, TrxA, DsbC, DsbA, IF2, Sumo, 3 fLag, Avi, KSI, CKS, SKP, BFR, GrPE, btuF and Ectin.
According to a second aspect of the present invention there is provided a DNA molecule comprising a DNA sequence encoding any one of the PTH fusion proteins described above.
Furthermore, the DNA molecule also comprises a connecting sequence and a connecting sequence, wherein the connecting sequence is respectively positioned at the 5 'end and the 3' end of the DNA sequence of the PTH fusion protein, the connecting sequence at the 5 'end is a 5' end enzyme cutting site, and the connecting sequence at the 3 'end is a stop codon and a 3' end enzyme cutting site which are sequentially connected; preferably, the 5 'end cleavage site is a BamHI cleavage site, the 3' end cleavage site is an XhoI cleavage site, and the stop codon is TAA.
According to a third aspect of the present invention there is provided a recombinant plasmid having operatively linked thereto a DNA molecule encoding any one of the above.
According to a fourth aspect of the present invention, there is provided an engineered bacterium comprising any one of the recombinant plasmids described above.
According to a fifth aspect of the present invention, there is provided a method for preparing a PTH fusion protein, the method comprising: artificially synthesizing a DNA sequence shown as SEQ ID NO. 2; connecting the DNA sequence to an expression vector with a tag sequence to construct a recombinant plasmid; screening positive recombinant plasmids, and transforming the positive recombinant plasmids into host cells to obtain transformed cells; and inducing the transformed cell to express to obtain the PTH fusion protein.
Further, the expression vector with the tag sequence is selected from any one of the following: pET-SKP, pET-28a, pET-MBP, pET-NusA, pET-TF, pET-MysB, pET-GST, pET-21a, pET-TrXA, pET-DsbC, pET-DsbA, pET-IF2, pET-Sumo, pET-3 fLag, pET-Avi, pET-KSI, pET-CKS, pET-BFR, pET-GrPE, pET-btuF and pET-Ectin.
According to a sixth aspect of the present invention, there is provided a PTH detection reagent comprising any one of the PTH fusion proteins described above.
Further, the detection reagent is a calibration product or a quality control product.
Further, the detection reagent further comprises a stabilizing solution for stabilizing the fusion protein, preferably the stabilizing solution comprises: a base component comprising a phosphate buffer at a pH of 7.2 to 7.5; and a stabilizing component comprising at least one of animal serum, a saccharide, a penetrant, and a detergent; preferably, the animal serum is selected fromAt least one of goat serum, bovine serum and horse serum, sugar is selected from at least one of sucrose, trehalose, fructo-oligosaccharide and dextran, penetrating agent is selected from at least one of glycine, arginine and proline, and detergent is selected from at least one of cholic acid, deoxycholic acid, sodium dodecyl sulfate, tween 20 and tween 80; more preferably, the base component comprises: 20-100 mM phosphate buffer solution, 50-100 mM NaCl, 0.01-1% chelating agent and 0.01-1% preservative, wherein the chelating agent is EDTA or EGTA, and the preservative is NaN3Or
300, respectively; more preferably, the stabilizing component comprises 1-20 wt% of fetal calf serum, 0.1-2M of glycine, 0.01-0.2 wt% of tween and 0.5-8 wt% of sucrose or trehalose; further preferably, the stabilizing component comprises 2-10 wt% of fetal calf serum, 0.2-1M of glycine, 0.01-0.1 wt% of tween and 1-5 wt% of sucrose or trehalose.
According to a seventh aspect of the present invention, there is provided a PTH detection kit comprising any one of the PTH detection reagents described above.
According to an eighth aspect of the present invention, there is provided a use of any one of the PTH fusion proteins described above, any one of the PTH detection reagents described above, or any one of the kits described above in clinical assay of PTH antigen.
Further, the application includes: and taking the PTH detection fusion protein as a calibration product or a quality control product, and carrying out clinical detection on the PTH antigen through a semi-automatic immunoassay analyzer or a full-automatic immunoassay analyzer.
By applying the technical scheme of the invention, the full-length PTH fusion protein with the tag sequence can provide a more comprehensive binding region when being bound with the antibody, thereby improving the detection accuracy. On the basis, in the preferred embodiment of the present application, the stable solution of the full-length PTH fusion protein is obtained by selecting a specific kind of stable solution and screening by optimizing the content ratio of the components. The PTH fusion protein can exist in a liquid state at normal temperature and can stably exist for a long time in the stabilizing solution, so that the accuracy of clinical detection and detection is improved, and the convenience of detection is improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows the degradation rate of NusA-PTH (1-84) fusion protein in stable A, B, C, D, E, F as a function of time in example 5 of the present application;
FIG. 2 shows the degradation rate of the DsbA-PTH (1-84) fusion protein in stable G, H, I, J, K, L as a function of time in example 6 of the present application;
FIG. 3 shows the degradation rate of TF-PTH (1-84) fusion protein in stabilizing solution M as a function of time in example 7 of the present application.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background, PTH in normal humans is released into the blood from the parathyroid gland and subsequently undergoes cleavage into various fragments. The fragment forms in serum are mainly PTH (35-84) and PTH (7-84). The current test sample is usually the venous blood of the arm, and the test method is usually a double antibody sandwich method. Because there are many forms of PTH fragments, one or more fragments may be involved in the mab-binding region used to detect PTH. However, the fusion protein of PTH 1-34 in the prior art cannot meet the requirements of serving as a calibration product or a quality control product, and the area where the fusion protein can be combined with a monoclonal antibody is limited when PTH is detected, so that certain influence is bound to the accuracy of clinical detection.
In order to ameliorate the above-mentioned disadvantages of the prior art, in one exemplary embodiment of the present application, there is provided a PTH fusion protein comprising: a tag sequence; the full-length amino acids at positions 1-84 of PTH, wherein the tag sequence is located at the C-terminus or N-terminus of the full-length amino acids at positions 1-84 of PTH.
The PTH fusion protein provided by the application reserves the full-length sequence of the PTH protein on one hand, can provide a more comprehensive binding region when being bound with an antibody, and further improves the detection accuracy; on the other hand, a proper tag sequence is selected according to actual needs, which is beneficial to purification of the fusion protein and enables the expression quantity and activity of the fusion protein to be relatively higher.
In the above PTH fusion protein, the tag sequence can be selected from the existing protein tags. In a preferred embodiment of the present application, the tag sequence is selected from any one of the following: MBP, NusA, TF, MysB, GST, T7, 6 His + T7, TrXA, DsbC, DsbA, IF2, Sumo, 3 fLag, Avi, KSI, CKS, SKP, BFR, GrPE, btuF and Ectin.
More preferably, the tag sequence is selected from the group consisting of NusA, TF, TrXA, DSbC, DsbA, CKS and SKP, which has advantages over other tag sequences in three respects: (1) the stability of the label peptide fragment is good; (2) the tag peptide fragment can promote the correct folding of the target protein structure, such as in the form of molecular chaperones or disulfide bonds; (3) the label peptide segment has the effects of easy expression and strong dissolving-assisting effect, and is beneficial to improving the expression efficiency of the target protein. The fusion protein adopting the tag sequences has higher yield after expression and purification.
In a second exemplary embodiment of the present application, a DNA molecule is provided, which comprises a DNA sequence encoding any one of the PTH fusion proteins described above.
Depending on the requirements of the plasmid used for the specific expression of the above fusion protein, the 5 'and 3' ends of the DNA sequence encoding the full length amino acids of PTH1-84 can be appropriately modified to allow for proper expression in the plasmid. The modification comprises adding enzyme cutting sites at the 5 'end and the 3' end so as to cut the full-length amino acid sequence of PTH1-84 for utilization when needed. The manner in which a particular fusion protein is linked to a plasmid may be determined on a case-by-case basis, and includes, but is not limited to, enzymatic ligation, such as PCR ligation.
In a preferred embodiment, the DNA molecule further comprises a linker sequence located at the 5 'end and a linker sequence located at the 3' end of the DNA sequence of the PTH fusion protein, respectively, i.e. (the DNA molecule comprises, in order from the 5 'end to the 3' end, a 5 'linker sequence-tag sequence-DNA sequence-3' linker sequence, or a 5 'linker sequence-DNA sequence-tag sequence-3' linker sequence), wherein the 5 'linker sequence is a 5' cleavage site, and the 3 'linker sequence comprises, in order, a stop codon and a 3' cleavage site; preferably, the 5 'end cleavage site is a BamHI cleavage site, the 3' end cleavage site is an XhoI cleavage site, and the stop codon is TAA. The existence of the above-mentioned linker sequence facilitates the cleavage of PTH (1-84) from the fusion protein for practical use as desired.
In a third exemplary embodiment of the present application, there is provided a recombinant plasmid having a DNA molecule encoding any one of the above operably linked thereto.
In a fourth exemplary embodiment of the present application, an engineered bacterium is provided, which comprises any one of the recombinant plasmids described above.
The DNA molecule, the recombinant plasmid and the engineering bacteria can all express to obtain the full-length PTH fusion protein, have the advantages of improving the detection accuracy and stability when being used as a PTH standard product, and can accurately detect the stability of used instruments or reagents when being used as a quality control product.
In a fifth exemplary embodiment of the present application, there is provided a method for preparing a PTH fusion protein, the method comprising: artificially synthesizing a DNA sequence shown as SEQ ID NO. 2; connecting the DNA sequence to an expression vector with a tag sequence to construct a recombinant plasmid; screening positive recombinant plasmids, and transforming the positive recombinant plasmids into host cells to obtain transformed cells; and inducing the transformed cell to express to obtain the PTH fusion protein.
The sequence shown in SEQ ID NO. 2 is a DNA sequence of a full-length amino acid sequence of the coding PTH1-84 and a connecting sequence, and is connected with a series of expression vectors with different tag sequences to obtain a series of recombinant plasmids. And then a series of PTH fusion proteins with different tag sequences can be obtained through the conventional steps of screening positive recombinant plasmids and inducing expression.
Similar to the conventional method, the above preparation method requires codon optimization of the DNA sequence encoding the full-length amino acid sequence of PTH1-84 according to the type of the host cell to be expressed and its codon preference before artificially synthesizing the sequence represented by SEQ ID NO. 2.
In the above preparation method, the vector having a tag sequence can be selected appropriately from existing vectors having tag sequences as required. In a preferred embodiment of the present application, the expression vector with the tag sequence is selected from any one of the following: pET-SKP, pET-28a, pET-MBP, pET-NusA, pET-TF, pET-MysB, pET-GST, pET-21a, pET-TrXA, pET-DsbC, pET-DsbA, pET-IF2, pET-Sumo, pET-3 fLag, pET-Avi, pET-KSI, pET-CKS, pET-BFR, pET-GrPE, pET-btuF and pET-Ectin. The expression vector with the tag sequence is helpful for improving the expression amount and/or activity of the expressed fusion protein.
More preferably, the expression vector having the tag sequence is selected from any one of pET-NusA, pET-TF, pET-TrxA, pET-DSbC, pET-DsbA, pET-CKS and pET-SKP. Compared with other tag sequences, the tag sequence has the following advantages in three aspects: (1) the stability of the label peptide fragment is good; (2) the tag peptide fragment can promote the correct folding of the target protein structure, such as in the form of molecular chaperones or disulfide bonds; (3) the label peptide segment has the effects of easy expression and strong dissolving-assisting effect, and is beneficial to improving the expression efficiency of the target protein.
In a sixth exemplary embodiment of the present application, there is provided a PTH detection reagent comprising any one of the PTH fusion proteins described above. The detection reagent containing the PTH fusion protein has the full-length sequence of the PTH protein, and can provide a more comprehensive binding region when being bound with an antibody, thereby improving the detection accuracy.
The PTH detection reagent can be used as a calibration product or a quality control product according to the actual application requirements. In a preferred embodiment of the present application, the detection reagent is a calibrator or a quality control reagent. Need toIt is to be noted that, in the case of a detection reagent specifically used as a calibrator, a quality controller or the like, the above-mentioned PTH fusion protein is usually present in a protein buffer capable of maintaining its activity or facilitating the detection of its activity, such as a phosphate buffered saline (PBS buffer). In a specific use state, the PTH fusion protein is present in 50mM PBS buffer (formulation: 100mM NaCl, 0.1% EDTA-2K, 0.09% NaN)350mM phosphate buffer, pH7.4), in which the presence of the PTH fusion protein enables instant detection.
In order to further improve the stability of the PTH detecting reagent in a storage state and to improve the convenience of use. In a preferred embodiment of the present application, the detection reagent further comprises a stabilizing solution for stabilizing the fusion protein. Any component capable of enhancing the stability of the buffer solution capable of realizing the detection of the PTH fusion protein can be used as a component of the stable solution of the detection reagent of the present application.
In another preferred embodiment of the present application, the stabilizing liquid comprises: a base component comprising a phosphate buffer at a pH of 7.2 to 7.5; and a stabilizing component, wherein the stabilizing component comprises at least one of animal serum, osmotic agent, saccharide, and detergent. The application verifies through a large number of experiments that the storage stability of the full-length PTH fusion protein can be improved by adding at least one component on the basis of the existing protein buffer solution, such as phosphate buffer solution.
Preferably, the animal serum is at least one selected from goat serum, bovine serum and horse serum, the saccharide is at least one selected from sucrose, trehalose, fructo-oligosaccharide and dextran, the penetrating agent is at least one selected from glycine, arginine and proline, and the detergent is at least one selected from cholic acid, deoxycholic acid, sodium dodecyl sulfate, tween 20 and tween 80. Among them, fructo-oligosaccharide and dextran with polymerization degree of 3-4 and molecular weight of 504-667 are preferred.
In a more preferred embodiment of the present application, the above-mentioned base component comprises: 20 to 100mM phosphate buffer, 50 to 100mM NaCl, 0.01 to 1% EDTA (by ED)TA-2K, dipotassium ethylene diamine tetraacetate, or EDTA-2Na, in the form of disodium ethylene diamine tetraacetate) and 0.01-0.01% of NaN3Or
300. In a more preferred embodiment of the present application, the stabilizing component comprises 1-20% of fetal bovine serum, 0.1-2M glycine, 0.01-0.2% of Tween and 0.5-8% of sucrose or trehalose; further preferably, the stabilizing component comprises 2-10% of fetal calf serum, 0.2-1M of glycine, 0.01-0.1% of tween and 1-5% of sucrose or trehalose. The above Tween is preferably Tween 20 and Tween 80.
In the stabilizing solution,% represents mass percentage, and M represents the final concentration of the component in the stabilizing solution, namely mol/L. It should be noted that% represents the mass percentage throughout the specification. Furthermore, hereinafter, the stabilizing solution M means the Mth stabilizing solution, and M represents the final concentration of the component in the stabilizing solution, i.e., mol/L, elsewhere.
In the preferred embodiment, the PTH fusion protein can be stably present in the stabilizing solution not only in a liquid form at normal temperature but also for a long period of time by selecting a specific kind of stabilizing solution and optimizing the content ratio between the components. The method not only improves the detection accuracy in clinical detection, but also improves the detection convenience.
In the preferred embodiment, the stabilizing solution used for the PTH fusion protein is slightly different depending on the label attached to the PTH fusion protein. In a more preferred embodiment, the NusA-PTH (1-84) fusion protein has the basic components in a stabilizing solution (pH7.4) of: 50mM phosphate buffer, 100mM NaCl, 0.1% EDTA-2K, and 0.09% NaN3(ii) a The stabilizing components comprise 1-20% of fetal calf serum, 0.1-2M of glycine, 0.05% of Tween 20 and 5% of sucrose or trehalose. Further preferably, for the NusA-PTH (1-84) fusion protein, the basic components in the stabilizing solution are: 50mM phosphate buffer, 100mM NaCl, 0.1% EDTA-2K, and 0.09% NaN3(ii) a The stabilizing component is 2% or 10% fetal calf serum, 0.2M or1M glycine, 0.05 % tween 20 and 5% sucrose or trehalose.
In another preferred embodiment, the DsbA-PTH (1-84) fusion protein has the following basic components in a stabilizing solution (pH 7.4): 50mM phosphate buffer, 100mM NaCl, 0.1% EDTA-2K and
300, respectively; the stabilizing component comprises 5% of fetal calf serum, 0.5M of glycine, 0.01-0.2% of Tween 20 and 0.5-8% of sucrose or trehalose. Further preferably, the DsbA-PTH (1-84) fusion protein has the basic components in a stabilizing solution (pH7.4) of: 50mM phosphate buffer, 100mM NaCl, 0.1% EDTA-2K and
300, respectively; the stabilizing component is 5% fetal calf serum, 0.5M glycine, 0.05% or 0.1% Tween 20 and 1% or 5% sucrose or trehalose.
In a preferred embodiment, the optimal stabilizing solution for the TF-PTH (1-84) fusion protein comprises the following basic components: 50mM phosphate buffer, 100mM NaCl, 0.1% EDTA-2K, and 0.09% NaN3(ii) a The stabilizing component is 10% fetal calf serum, 1M glycine, 0.05 % Tween 20 and 5% sucrose or trehalose.
In a preferred embodiment, the optimal stabilizing solution for the TrxA-PTH (1-84) fusion protein comprises the following basic components: 50mM phosphate buffer, 100mM NaCl, 0.1% EDTA-2K, and 0.09% NaN3(ii) a The stabilizing component is 8% fetal calf serum, 0.5M glycine, 0.05% Tween 20 and 3% sucrose or trehalose.
In a seventh exemplary embodiment of the present application, there is provided a PTH detection kit comprising any one of the PTH detection reagents described above. The kit containing the PTH detection reagent has the advantages of convenience and rapidness in detection and high accuracy.
In an eighth exemplary embodiment of the present application, there is provided a use of any one of the PTH fusion proteins, any one of the PTH detection reagents, or any one of the kits described above in clinical detection of PTH antigen. In a preferred embodiment of the present application, the application comprises: and taking the PTH detection fusion protein as a calibration product or a quality control product, and carrying out clinical detection on the PTH antigen through a semi-automatic immunoassay analyzer or a full-automatic immunoassay analyzer. The calibration article is composed of a series of reference articles of known concentration and is used to provide a standard curve for the test agent, and different reference articles are used to prepare standard curves with different sensitivities and stabilities. The application of the PTH fusion protein as the calibration product has the advantages of high detection sensitivity and high accuracy. And the quality control product is a reference product of known concentration specially used for quality control purposes. The PTH fusion protein is applied as a quality control product, so that the accuracy and stability of monitoring and calibration are higher.
The advantageous effects of the present application will be described in detail below with reference to specific examples.
Method for detecting raw material source and fusion protein
1 materials of the experiment
The PTH gene sequence was synthesized by Nanjing Kinshire.
2 Strain vector
Coli (E.coLi) DH5 alpha competent cells, tagged prokaryotic series expression vectors (pET-28a, pET-MBP, pET-NusA, pET-TF, pET-MysB, pET-GST, pET-21a, pET-TrXA, pET-DSbC, pET-DSbA, pET-IF2, pET-Sumo, pET-3 flag, pET-Avi, pET-KSI, pET-CKS, pET-BFR, pET-GrPE, pET-btuF, pET-otecin), prokaryotic expression host bacteria BL21(DE3) competent cells, provided by Snibe preservation.
3 Experimental test solution, medicine and main equipment
PCR Mix and DNA Marker were purchased from Beijing holotype gold organism; t is4DNA ligase, endonucleases BamHI and XhoI, vector universal upstream and downstream primers T7 and T7ter were purchased from thermo; DNA extraction test solution boxes, gel recovery test solution boxes and plasmid extraction test solution boxes used in DNA manipulation were purchased from Omega; ampicillin (Amp), kanamycin (Kan), IPTG, agarose and agar powder were purchased from Shanghai.
PTH1-84 (WHO international standard), NIBSC code: 95/646, available from the world health organization BioStandard International laboratory.
4 fusion expression PTH (1-84) detection method
A detection instrument: shenzhen city new industry biomedical engineering shares GmbH Maglumi 4000P
The detection method comprises the following steps: detecting PTH concentration by chemiluminescence immune sandwich method, adopting one monoclonal antibody of PTH to mark ABEI, another to mark FITC, sample, standard solution and ABEI marked monoclonal antibody, FITC marked monoclonal antibody, mixing uniformly, incubating for 15 min at 37 deg.C to form sandwich, adding magnetic microsphere coated with goat anti-FITC antibody, incubating for 5 min at 37 deg.C, adding magnetic field for precipitation, removing supernatant, washing precipitation complex with washing solution for 2 times, directly entering sample measuring chamber, and automatically pumping luminescent substrates 1(NaOH) and 2 (H) into the instrument2O2) The relative light intensity (RLU) emitted within 3 seconds is automatically monitored. PTH concentration and RLU become certain proportional relation, and the instrument is automatic to be fit and calculates PTH concentration.
Detecting a test solution: serum parathyroid hormone test kit (chemiluminescence method) provided in each of the following examples or comparative examples.
The linear range of the PTH (1-84) detected by the test solution kit is 0-5000 pg/mL.
(II) examples and comparative examples
Example 1
(I) construction of recombinant cloning plasmid
Design and Artificial Synthesis of DNA sequence expressing PTH (1-84): according to the amino acid sequence of PTH (1-84) (see SEQ ID NO:1), the cDNA sequence of the artificially synthesized protein coding for the human parathyroid hormone 1-84 is shown as SEQ ID NO:2, and the DNA sequence which is artificially synthesized and optimized and is suitable for the expression of the Escherichia coli is shown as SEQ ID NO:3 according to the codon preference of the Escherichia coli. When PTH (1-84) gene is synthesized, BamHI cleavage sites are introduced at the 5 'end of the gene and termination codons TAA and XhoI cleavage sites are introduced at the 3' end.
To facilitate subsequent subcloning, the synthetic gene was cloned into a pUC57-simple plasmid for the purpose of preserving the synthetic DNA sequence. Plasmid pUC57-simple contains BamHI and XhoI cleavage sites.
(II) construction of recombinant expression plasmid and recombinant engineering bacteria:
1. preparing a target gene fragment
Extracting pUC57-PTH plasmid containing PTH (1-84) coding sequence with DNA extracting kit, double digestion with BamHI/XhoI, separating small fragment in 1% agarose gel electrophoresis, cutting gel containing about 11KD fragment, recovering about 11KD fragment with gel DNA recovering kit, and electrophoretic checking.
2. Preparation of expression vector fragments
The pET28a (+) plasmid DNA was extracted using a DNA extraction kit, the large fragment was separated by electrophoresis using BamHI/XhoI, 1% agarose gel, the gel containing the large fragment was cut off, and the large fragment was recovered using a gel DNA recovery kit and kept for later use after electrophoretic verification.
The remaining expression plasmids pET-28a (+), pET-MBP, pET-NusA, pET-TF, pET-MysB, pET-GST, pET-21a, pET-TrXA, pET-DsbC, pET-DsbA, pET-IF2, pET-Sumo, pET-3 fLag, pET-Avi, pET-KSI, pET-CKS, pET-BFR, pET-GrPE, pET-btuF, and pET-Ectin were recovered in the same manner as described above.
3. Construction of recombinant expression plasmid pET-PTH (1-84)
The target gene fragment prepared in the above steps 1 and 2 and the DNA fragment of the expression vector fragment were mixed uniformly in different molar ratios (2/8, 3/7, 4/6, 5/5), ligated with T4 DNA ligase at 16 ℃ for 30min, the ligation product was transformed into E.coli DH 5. alpha. competent cells, and the transformed competent cells were spread on agarose plates (1% peptone, 0.5% yeast extract, 1% NaCl and 2% agar) containing the respective resistances, and cultured overnight at 37 ℃.
4. Positive recombinants screening
Resistant single colonies (i.e., white spots) were randomly picked from the above transformation plates using sterile toothpicks and identified by PCR. And (3) inoculating the single colony which is positive in PCR verification into 5mL of LB/antibiotic culture solution, extracting plasmids, performing enzyme digestion identification, and performing 1% agarose gel electrophoresis to identify the recon.
And (3) selecting a plasmid of which the PCR and the double enzyme digestion are positive clones for sequencing, and committing the sequencing work to Thermo company for completion. The sequencing results were subjected to BLAST analysis to confirm that the DNA fragments had been ligated to the corresponding vectors, and the correct recombinant plasmid was designated pET-PTH (1-84). The recombinant plasmid pET-PTH (1-84) DNA is used for transforming Escherichia coli BL21(DE3), and the obtained recombinant engineering bacteria is the recombinant engineering bacteria for expressing rPTH (1-84) fusion protein.
Example 2 inducible expression of recombinant engineered bacteria
8 single colonies were picked, and positive single colonies were aseptically transferred to 50mL centrifuge tubes containing 10mL LB broth (containing resistance) and incubated overnight (about 15 hours) at 37 ℃ on a shaker at 220 rpm. Transferred to a 1L conical flask containing 350mL of LB medium (containing resistance) in a volume of 1/100, and subjected to amplification culture in a constant temperature oscillator at 37 ℃ and 250 rpm; OD600When the concentration reaches 0.6, IPTG is added to the final concentration of 0.3Mm, induction expression is carried out at 20 ℃ and 180rpm, induction culture is carried out for 20h, then bacteria are collected by centrifugation, and centrifugation is carried out for 10 min at 4 ℃ and 10000 rpm. Collecting the thallus precipitate, and storing in a refrigerator at-40 deg.C for use.
Example 3
As in examples 1 and 2, expression vectors having protein tags such as NusA, TF, TrxA, DsbA, DsbC, CKS, SKP, etc. were fusion-expressed together with the target protein PTH (1-84), respectively, to obtain fusion proteins such as NusA-PTH (1-84), TF-PTH (1-84), TrxA-PTH (1-84), DsbA-PTH (1-84), DsbC-PTH (1-84), CKS-PTH (1-84), and SKP-PTH (1-84), respectively, and then PBS buffer (formulation: 100mM NaCl, 0.1% EDTA-2K, 0.09% NaN, respectively, was added350mM phosphate buffer, pH7.4) the prepared fusion protein containing PTH (1-84) was diluted 1000-fold as a stock solution. And then diluting the prepared PTH (1-84) stock solution and the diluent according to the volume ratio of 1:1, 1:3, 1:7, 1:15 and 1:31 respectively, determining the concentration of the stock solution and comparing the concentration with a PTH (1-84) WHO international standard, and finally calculating the original concentration of each fusion expression PTH (1-84) according to the dilution ratio, namely the yield. The results of the measurements are shown in Table 1 below.
TABLE 1 PTH (1-84) fusion protein yield
Note: the linear range of the serum parathyroid hormone detection test solution box (chemiluminescence method) is 0-5000 pg/mL, and when the concentration of a detection sample is greater than 5000pg/mL, the result is shown to be greater than 5000 pg/mL.
Table 1 the results show that: the yield of the prepared PTH (1-84) fusion protein is 45-65 mg/mL, wherein the higher yield is NusA-PTH (1-84) and DsbA-PTH (1-84).
Example 4
Under the same conditions, recovery experiments of PTH (1-84) WHO international standard and PTH (1-84) fusion protein prepared by the present invention were carried out. The specific operation is as follows:
known concentrations of PTH (1-84) WHO international standard and PTH (1-84) fusion protein prepared according to the present invention were mixed with PBS buffer (formulation: 100mM NaCl, 0.1% EDTA-2K, 0.09% NaN)350mM phosphate buffer, pH7.4) to 5000pg/mL, defined as J-point concentration, diluting a J-point concentration PTH (1-84) WHO international standard and a J-point concentration PTH (1-84) fusion protein to PBS buffer at a volume ratio of 1:1 to obtain I-point concentration, and obtaining H-point concentration, G-point concentration, F-point concentration, E-point concentration, D-point concentration, C-point concentration and B-point concentration according to the same dilution ratio, wherein A-point concentration is the PTH concentration in PBS buffer, namely PTH concentration is 0. The concentration of each spot was measured under the same conditions. The results of the measurements are shown in Table 2 below.
Table 2.
Table 2 the results show: the fusion protein prepared according to the present invention further includes PTH (1-84) as determined by dilution of the fusion-expressed PTH (1-84) with the PTH (1-84) WHO standard in a ratio such that the measured concentration substantially corresponds to the target concentration.
Example 5
Respectively preparing PTH standard substance stabilizing solutions A, B, C, D, E and F according to a formula shown in a table 3, preparing PTH fusion protein according to the prokaryotic PTH expression method, respectively diluting NusA-PTH (1-84) fusion protein into 1500pg/mL by using PTH standard substance stabilizing solutions A, B, C, D, E and F, averagely dividing the diluted standard substance solution into 9 parts, placing the parts in a dark place at 40 ℃, taking 1 part every 3-4 days to detect the PTH concentration according to the method, and drawing a curve of PTH degradation rate changing along with time.
The detection result is shown in fig. 1, the change curve of the degradation rate of the PTH along with the time reflects the stability of the PTH standard, and as can be seen from fig. 1, the stabilizing solution has a good protection effect on the fusion expressed PTH (1-84) standard. Wherein, when the concentration of fetal calf serum in the standard substance stabilizing solution is 2-10% and the concentration of glycine is 0.2-1M (namely stabilizing solutions B and E), the stabilizing effect of the stabilizing solution on the fusion protein is better.
TABLE 3 PTH stabilizer A, B, C, D, E, F composition
Example 6
Separately, a stable solution G, H, I, J, K, L was prepared according to the formulation of Table 4, and PTH fusion proteins were prepared according to the method for prokaryotic PTH expression of the present application. Respectively diluting DsbA-PTH (1-84) fusion protein to 1500pg/mL by using a PTH standard substance stabilizing solution G, H, I, J, K, L, averagely dividing the diluted standard substance solution into 9 parts, placing the solution at 40 ℃ in a dark place, taking 1 part every 3-4 days, detecting the PTH concentration, and drawing a PTH degradation rate change curve along with time.
The detection result is shown in fig. 2, the change curve of the degradation rate of the PTH along with the time reflects the stability of the PTH standard, and it can be seen that the stabilizing solution of the present application has a good protective effect on the fusion-expressed PTH (1-84) standard. Wherein, when the concentration of the saccharides in the standard substance stabilizing solution is 1 to 5 percent and the concentration of the washing solution is 0.02 to 0.1 percent (namely stabilizing solutions H and J), the degradation rate of the PTH is lower.
TABLE 4 stabilizing solution formulations of examples 6-9
Example 7
PTH stabilizer M was prepared according to the formulation in Table 4 above, as follows:
the preparation method of the 20mL PTH standard substance stabilizing solution M comprises the following steps: 1mL of 1M sodium phosphate buffer and 8mL of purified water were added to the beaker, mixed, and the pH was adjusted to 7.4. + -. 0.1. Then 0.117g of sodium chloride, 1g of sucrose, 1.501g of glycine and 0.02g of EDTA were added and mixed. Then 0.1mL of 10% tween 20 solution was added and mixed. Next, 2mL of fetal bovine serum was added and mixed, 0.2mL of 9% sodium azide, and the pH of the solution was adjusted to 7.4. + -. 0.1 again. Finally, the volume is adjusted to 20mL by purified water.
Fusion expressed PTH fusion proteins were prepared according to the prokaryotic PTH expression method of the present application using common PBS buffer (formulation: 100mM NaCl, 0.1% EDTA-2K, 0.09% NaN)350mM phosphate buffer solution, pH7.4) and the PTH standard substance stabilizing solution M of the application dilute TF-PTH (1-84) fusion protein expressed by fusion to 1500pg/mL, each group of diluted standard substance solution is averagely divided into 9 parts, the diluted standard substance solution is placed in a dark place at 40 ℃, 1 part of the diluted standard substance solution is taken every 3-4 days to detect the PTH concentration by the method, and a PTH degradation rate change curve along with time is drawn.
The detection result is shown in fig. 3, the change curve of the degradation rate of the PTH along with the time reflects the stability of the PTH standard, and the result shows that the stability of the fusion-expressed PTH in the stabilizing solution M is obviously better than that in the common buffer solution.
Example 8
PTH stabilizer N was prepared according to the formulation of Table 4 in the same manner as in example 7. The PTH fusion protein is prepared according to the prokaryotic PTH expression method, TrxA-PTH (1-84) fusion protein expressed by fusion of a PTH standard substance stabilizing solution N is respectively diluted into 50pg/mL, 1500pg/mL and 2500pg/mL, the diluted standard substance solution is divided into two groups, 9 parts are respectively arranged in each group, one group is placed in a dark place at the temperature of 2-8 ℃, and 1 part is taken every 1-4 months for detecting the PTH concentration. And the other group is placed in a dark place at 40 ℃, and 1 part of PTH is taken every 3-7 days for detecting the PTH concentration. The results of the measurements are shown in tables 5 and 6 below.
TABLE 5.2-8 deg.C detection results
TABLE 6.40 ℃ measurement results
As can be seen from tables 5 and 6, fusion-expressed TrxA-PTH was added to the liquid stabilizing solution of the present application:
the fusion-expressed PTH standard can be stably stored for at least 30 days at 40 ℃;
the fusion expressed PTH standard can be stably stored for at least 12 months at the temperature of 2-8 ℃.
In addition, the inventors studied the effect of each component in the stabilizing solution on the stability of the NusA-PTH (1-84) fusion protein separately, in the same manner as in example 5, and the specific formulation of each stabilizing solution is shown in Table 7 below.
Table 7:
preparing PTH standard substance stabilizing solutions 1 to 12 according to a formula shown in Table 7, diluting NusA-PTH (1-84) fusion protein to 1500pg/mL by using the PTH standard substance stabilizing solutions 1 to 12 respectively, diluting the NusA-PTH (1-84) fusion protein to 1500pg/mL by using a common buffer solution as a contrast, averagely dividing the diluted standard substance solution into 9 parts, placing the parts in a dark place at 40 ℃, taking 1 part every 3 to 4 days to detect the PTH concentration according to the method, and drawing a curve of PTH degradation rate changing along with time.
The test results are shown in Table 8, and Table 8 shows the degradation rate of the NusA-PTH (1-84) fusion protein with time, reflecting the effect of the stabilizing solution with different components on maintaining the stability of the PTH standard. As can be seen from Table 8, compared with the common buffer solution formed by only basic components, the stabilizing solutions 1 to 12 of the present application have good protective effect and better stabilizing effect on the fusion expressed NusA-PTH (1-84) standard.
Moreover, as can be seen from table 8, the stabilizing solutions 6, 7, 8 and 9 of the standard substances can obtain better stabilizing effect than the common buffer solution by adding only one of animal serum, penetrant, saccharide and detergent on the basis of the components of the common buffer solution. In the case of adding any two of these, stabilizing solutions 5 and 10, the stability of the fusion protein was more effective. When three kinds are added, the stabilizing effect of the stabilizing solution on the fusion protein is better than that of one or two kinds of the stabilizing solution. And the stabilizing solution containing the stabilizing component has the best stabilizing effect on the fusion protein. Wherein, the effect of the stabilizing liquid when the animal serum is fetal calf serum, the saccharide is sucrose/trehalose, the penetrating agent is glycine, and the detergent is Tween is relatively better than the effect of the stabilizing liquid when the animal serum is goat or horse serum, the saccharide is fructo-oligosaccharide/dextran, the penetrating agent is arginine/proline, and the detergent is cholic acid, deoxycholic acid, and sodium dodecyl sulfate.
Table 8:
attached: ctr in the table above represents the common buffer.
From the above description, it can be seen that the above embodiments of the present application provide a more comprehensive binding region when binding to an antibody by using a full-length PTH fusion protein having a tag sequence, thereby improving detection accuracy. And selecting a specific type of stable solution, and screening to obtain the stable solution of the full-length PTH fusion protein by optimizing the content ratio of the components. The PTH fusion protein can exist in a liquid state at normal temperature and can stably exist for a long time in the stabilizing solution, so that the accuracy of clinical detection and detection is improved, and the convenience of detection is improved.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Shenzhen City New industry biomedical engineering shares Limited
<120> PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application
<130> PN82165SZSW
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 84
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 1
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser
35 40 45
Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu
50 55 60
Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys
65 70 75 80
Ala Lys Ser Gln
<210> 2
<211> 252
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 2
tctgtgagtg aaatacagct tatgcataac ctgggaaaac atctgaactc gatggagaga 60
gtagaatggc tgcgtaagaa gctgcaggat gtgcacaatt ttgttgccct tggagctcct 120
ctagctccca gagatgctgg ttcccagagg ccccgaaaaa aggaagacaa tgtcttggtt 180
gagagccatg aaaaaagtct tggagaggca gacaaagctg atgtgaatgt attaactaaa 240
gctaaatccc ag 252
<210> 3
<211> 252
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 3
tctgtttctg aaatccagct gatgcacaac ctgggtaaac acctgaactc tatggaacgt 60
gttgaatggc tgcgtaaaaa actgcaggac gttcacaact tcgttgctct gggtgctccg 120
ctggctccgc gtgacgctgg ttctcagcgt ccgcgtaaaa aagaagacaa cgttctggtt 180
gaatctcacg aaaaatctct gggtgaagct gacaaagctg acgttaacgt tctgaccaaa 240
gctaaatctc ag 252
Claims (13)
1. A PTH detection reagent comprising a PTH fusion protein, wherein the PTH fusion protein comprises a tag sequence and the full-length amino acid sequence of PTH1 to 84,
wherein the tag sequence is located at the C-terminal or N-terminal of the full-length amino acid sequence of PTH1-84, and is selected from any one of the following: MBP, NusA, TF, MysB, GST, T7, 6 His + T7, TrxA, DsbC, DsbA, IF2, Sumo, 3 fLag, Avi, KSI, CKS, SKP, BFR, GrPE, btuF and Ectin;
the detection reagent is a calibration product or a quality control product, the detection reagent further comprises a stabilizing solution for stabilizing the fusion protein, and the stabilizing solution comprises:
a base component comprising a phosphate buffer at a pH of 7.2 to 7.5; and
a stabilizing component comprising at least two of animal serum, a saccharide, a penetrant, and a detergent.
2. The detection reagent of claim 1, wherein the PTH fusion protein is encoded by a DNA molecule, the DNA molecule further comprises a linker sequence located at the 5 'end and a linker sequence located at the 3' end of the DNA sequence of the PTH fusion protein, respectively, the linker sequence at the 5 'end is a 5' end enzyme cleavage site, and the linker sequence at the 3 'end is a stop codon and a 3' end enzyme cleavage site that are sequentially linked.
3. The detection reagent of claim 2, wherein the 5 'end cleavage site is a BamHI cleavage site, the 3' end cleavage site is an XhoI cleavage site, and the stop codon is TAA.
4. The detection reagent according to claim 2, wherein the DNA molecule is operably linked to a recombinant plasmid.
5. The detection reagent according to claim 4, wherein the recombinant plasmid is selected from any one of the following:
pET-SKP, pET-28a, pET-MBP, pET-NusA, pET-TF, pET-MysB, pET-GST, pET-21a, pET-TrXA, pET-DsbC, pET-DsbA, pET-IF2, pET-Sumo, pET-3 fLag, pET-Avi, pET-KSI, pET-CKS, pET-BFR, pET-GrPE, pET-btuF and pET-Ectin.
6. The detection reagent of claim 5, wherein the PTH fusion protein is obtained by transforming the recombinant plasmid and inducing expression of a host cell.
7. The detection reagent of claim 1, wherein the animal serum is at least one selected from goat serum, bovine serum and horse serum, the saccharide is at least one selected from sucrose, trehalose, fructo-oligosaccharide and dextran, the penetrant is at least one selected from glycine, arginine and proline, and the detergent is at least one selected from cholic acid, deoxycholic acid, sodium dodecyl sulfate, tween 20 and tween 80.
8. The detection reagent according to claim 1,
the base component comprises: 20-100 mM phosphate buffer solution, 50-100 mM NaCl, 0.01-1% chelating agent and 0.01-1% preservative, wherein the chelating agent is EDTA or EGTA, and the preservative is NaN3Or ProClin 300.
9. The detection reagent of claim 1, wherein the stabilizing component comprises 1-20 wt% of fetal bovine serum, 0.1-2M of glycine, 0.01-0.2 wt% of Tween and 0.5-8 wt% of sucrose or trehalose.
10. The detection reagent according to claim 9,
the stabilizing component comprises 2-10 wt% of fetal calf serum, 0.2-1M of glycine, 0.01-0.1 wt% of tween and 1-5 wt% of sucrose or trehalose.
11. A PTH detection kit comprising a PTH detection reagent according to any one of claims 1 to 10.
12. Use of a PTH detection reagent according to any one of claims 1 to 10 in the manufacture of a clinical PTH antigen detection kit.
13. The application according to claim 12, wherein the application comprises: and taking the PTH detection fusion protein as a calibration product or a quality control product, and carrying out clinical detection on the PTH antigen through a semi-automatic immunoassay analyzer or a full-automatic immunoassay analyzer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810127224.7A CN110128542B (en) | 2018-02-08 | 2018-02-08 | PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810127224.7A CN110128542B (en) | 2018-02-08 | 2018-02-08 | PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110128542A CN110128542A (en) | 2019-08-16 |
| CN110128542B true CN110128542B (en) | 2020-12-29 |
Family
ID=67567376
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810127224.7A Active CN110128542B (en) | 2018-02-08 | 2018-02-08 | PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110128542B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112143755A (en) * | 2020-10-19 | 2020-12-29 | 深圳市瑞普逊科技有限公司 | Human parathyroid hormone eukaryotic expression recombinant plasmid vector and construction method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807456B (en) * | 2005-01-19 | 2012-09-05 | 丁邦 | Recombinant human parathormone PTH1-34 preparation method |
| CN1861790A (en) * | 2006-04-25 | 2006-11-15 | 华东理工大学 | Preparation process of human recombined parathyroid hormone 1 84 |
| CN102304518A (en) * | 2011-08-15 | 2012-01-04 | 中国科学院上海生命科学研究院湖州工业生物技术中心 | Method for preparing human parathyroid hormone 1-34 |
| CN105353139A (en) * | 2015-09-28 | 2016-02-24 | 成都博奥新景医学科技有限公司 | Parathyroid hormone quantitative determination kit |
-
2018
- 2018-02-08 CN CN201810127224.7A patent/CN110128542B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110128542A (en) | 2019-08-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2316280A1 (en) | Novel g protein-coupled receptor | |
| CA2943228C (en) | Stabilized fibronectin based scaffold molecules | |
| Ito et al. | Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane | |
| CN110903383A (en) | Recombinant human type I collagen, encoding gene, engineering bacterium and application thereof | |
| KR20180087431A (en) | FKBP domain having a transglutaminase recognition site | |
| KR20220110556A (en) | Method for producing a peptide having physiological activity and a peptide comprising a single-chain linker | |
| Song et al. | Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model | |
| CN110128542B (en) | PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application | |
| Gram et al. | A novel approach for high level production of a recombinant human parathyroid hormone fragment in Escherichia coli | |
| JPH10512140A (en) | Assays, receptor proteins and ligands | |
| CN104610443A (en) | High-stability recombinant procalcitonin and preparation method and application thereof | |
| US4714674A (en) | Chemotactic assay for immunogenicity | |
| Liu et al. | Large scale preparation of recombinant human parathyroid hormone 1–84 from Escherichia coli | |
| CN107903307A (en) | A kind of high-affinity EDB FN targeting proteins peptides and its application | |
| JP5865002B2 (en) | Recombinant plasmid vector and protein production method using the same | |
| US6395875B1 (en) | Recombinant soluble adenovirus receptor | |
| EP1200588B1 (en) | Single-chain polypeptides comprising troponin i n-terminal fragments and troponin c | |
| CN112105635A (en) | Leader sequences for higher expression of recombinant proteins | |
| Fine et al. | Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization | |
| JPH06503711A (en) | Novel insulin-like growth factor binding protein (IGFBP-4) | |
| JP2001505764A (en) | Receptor binding assay, recombinant fusion receptor suitable for the receptor binding assay, vector for its preparation, and kit for performing the receptor binding assay | |
| CN101397562B (en) | Construction method for major histocompatibility complex/peptide tetramer for chicken and products thereof | |
| Käkönen et al. | Purification and characterization of recombinant osteocalcin fusion protein expressed inEscherichia coli | |
| JP4193499B2 (en) | Recombinant photoprotein and its complex | |
| JP2637392B2 (en) | Method for producing biologically active β-NGF by genetic manipulation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |